RESUMO
The HIV-1 capsid is composed of capsid (CA) protein hexamers and pentamers (capsomers) that contain a central pore hypothesised to regulate capsid assembly and facilitate nucleotide import early during post-infection. These pore functions are mediated by two positively charged rings created by CA Arg-18 (R18) and Lys-25 (K25). Here we describe the forced evolution of viruses containing mutations in R18 and K25. Whilst R18 mutants fail to replicate, K25A viruses acquire compensating mutations that restore nearly wild-type replication fitness. These compensating mutations, which rescue reverse transcription and infection without reintroducing lost pore charges, map to three adaptation hot-spots located within and between capsomers. The second-site suppressor mutations act by restoring the formation of pentamers lost upon K25 mutation, enabling closed conical capsid assembly both in vitro and inside virions. These results indicate that there is no intrinsic requirement for K25 in either nucleotide import or capsid assembly. We propose that whilst HIV-1 must maintain a precise hexamer:pentamer equilibrium for proper capsid assembly, compensatory mutations can tune this equilibrium to restore fitness lost by mutation of the central pore.
Assuntos
Proteínas do Capsídeo , Capsídeo , HIV-1 , Mutação , Montagem de Vírus , Replicação Viral , HIV-1/genética , HIV-1/fisiologia , Montagem de Vírus/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Capsídeo/metabolismo , Humanos , Replicação Viral/genética , Vírion/metabolismo , Vírion/genética , Células HEK293 , Infecções por HIV/virologia , Infecções por HIV/genéticaRESUMO
HIV-1 virion maturation is an essential step in the viral replication cycle to produce infectious virus particles. Gag and Gag-Pol polyproteins are assembled at the plasma membrane of the virus-producer cells and bud from it to the extracellular compartment. The newly released progeny virions are initially immature and noninfectious. However, once the Gag polyprotein is cleaved by the viral protease in progeny virions, the mature capsid proteins assemble to form the fullerene core. This core, harboring two copies of viral genomic RNA, transforms the virion morphology into infectious virus particles. This morphological transformation is referred to as maturation. Virion maturation influences the distribution of the Env glycoprotein on the virion surface and induces conformational changes necessary for the subsequent interaction with the CD4 receptor. Several host factors, including proteins like cyclophilin A, metabolites such as IP6, and lipid rafts containing sphingomyelins, have been demonstrated to have an influence on virion maturation. This review article delves into the processes of virus maturation and Env glycoprotein recruitment, with an emphasis on the role of host cell factors and environmental conditions. Additionally, we discuss microscopic technologies for assessing virion maturation and the development of current antivirals specifically targeting this critical step in viral replication, offering long-acting therapeutic options.
Assuntos
HIV-1 , Vírion , Montagem de Vírus , Replicação Viral , HIV-1/fisiologia , HIV-1/efeitos dos fármacos , Humanos , Vírion/metabolismo , Vírion/fisiologia , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , Fármacos Anti-HIV/farmacologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing virus that establishes life-long infection. KSHV is implicated in the etiology of Kaposi's sarcoma, and a number of rare hematopoietic malignancies. The present study focuses on the KSHV open reading frame 20 (ORF20), a member of the conserved herpesvirus UL24 protein family containing five conserved homology domains and a conserved PD-(D/E)XK putative endonuclease motif, whose nuclease function has not been established to date. ORF20 encodes three co-linear protein isoforms, full length, intermediate, and short, though their differential functions are unknown. In an effort to determine the role of ORF20 during KSHV infection, we generated a recombinant ORF20-Null KSHV genome, which fails to express all three ORF20 isoforms. This genome was reconstituted in iSLK cells to establish a latent infection, which resulted in an accelerated transcription of viral mRNAs, an earlier accumulation of viral lytic proteins, an increase in the quantity of viral DNA copies, and a significant decrease in viral yield upon lytic reactivation. This was accompanied by early cell death of cells infected with the ORF20-Null virus. Functional complementation of the ORF20-Null mutant with the short ORF20 isoform rescued KSHV production, whereas its endonuclease mutant form failed to enhance lytic reactivation. Complementation with the short isoform further revealed a decrease in cell death as compared with ORF20-Null virus. Finally, expression of IL6 and CXCL8, previously shown to be affected by the hCMV UL24 homolog, was relatively low upon reactivation of cells infected with the ORF20-Null virus. These findings suggest that ORF20 protein, with its putative endonuclease motif, promotes coordinated lytic reactivation for increased infectious particle production.
Assuntos
Herpesvirus Humano 8 , Fases de Leitura Aberta , Proteínas Virais , Ativação Viral , Humanos , Linhagem Celular , DNA Viral/genética , Regulação Viral da Expressão Gênica , Genoma Viral , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/metabolismo , Vírion/genética , Latência Viral , Replicação ViralRESUMO
Our examination of RNA helicases for effects on HIV-1 protein production and particle assembly identified Rocaglamide (RocA), a known modulator of eIF4A1 function, as an inhibitor of HIV-1 replication in primary CD4+ T cells and three cell systems. HIV-1 attenuation by low-nM RocA doses was associated with reduced viral particle formation without a marked decrease in Gag production. Rather, the co-localization of Gag and HIV-1 genomic RNA (gRNA) assemblies was impaired by RocA treatment in a reversible fashion. Ribonucleoprotein (RNP) immunoprecipitation studies recapitulated the loss of Gag-gRNA assemblies upon RocA treatment. Parallel biophysical studies determined that neither RocA nor eIF4A1 independently affected the ability of Gag to interact with viral RNA, but together, they distorted the structure of the HIV-1 RNP visualized by electron microscopy. Taken together, several lines of evidence indicate that RocA induces stable binding of eIF4A1 onto the viral RNA genome in a manner that interferes with the ordered assembly of Gag along Gag-gRNA assemblies required to generate infectious virions.
Assuntos
Benzofuranos , HIV-1 , RNA Viral , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , HIV-1/genética , Humanos , Replicação Viral/efeitos dos fármacos , RNA Viral/metabolismo , RNA Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Benzofuranos/farmacologia , Vírion/efeitos dos fármacos , Vírion/metabolismo , Linfócitos T CD4-Positivos/virologia , Montagem de Vírus/efeitos dos fármacos , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , Fator de Iniciação 4A em Eucariotos/metabolismo , Genoma Viral/efeitos dos fármacos , Ligação Proteica , Linhagem CelularRESUMO
Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0. We have demonstrated an essential role of VP0 E096 in VP0 cleavage independent of RNA encapsidation and generated a new model of capsid maturation, supported by bioinformatic analysis. This provides a molecular basis for RNA-dependence, where RNA induces conformational changes required for VP0 maturation, but that RNA packaging itself is not sufficient to induce maturation. These data have implications for understanding production of infectious virions and potential relevance for future vaccine and antiviral drug design.
Assuntos
Proteínas do Capsídeo , Montagem de Vírus , Montagem de Vírus/fisiologia , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Vírion/metabolismo , Enterovirus/fisiologia , Capsídeo/metabolismo , Infecções por Enterovirus/virologia , Infecções por Enterovirus/metabolismoRESUMO
The viral capsid performs critical functions during HIV-1 infection and is a validated target for antiviral therapy. Previous studies have established that the proper structure and stability of the capsid are required for efficient HIV-1 reverse transcription in target cells. Moreover, it has recently been demonstrated that permeabilized virions and purified HIV-1 cores undergo efficient reverse transcription in vitro when the capsid is stabilized by addition of the host cell metabolite inositol hexakisphosphate (IP6). However, the molecular mechanism by which the capsid promotes reverse transcription is undefined. Here we show that wild type HIV-1 virions can undergo efficient reverse transcription in vitro in the absence of a membrane-permeabilizing agent. This activity, originally termed "natural endogenous reverse transcription" (NERT), depends on expression of the viral envelope glycoprotein during virus assembly and its incorporation into virions. Truncation of the gp41 cytoplasmic tail markedly reduced NERT activity, suggesting that gp41 licenses the entry of nucleotides into virions. By contrast to reverse transcription in permeabilized virions, NERT required neither the addition of IP6 nor a mature capsid, indicating that an intact viral membrane can substitute for the function of the viral capsid during reverse transcription in vitro. Collectively, these results demonstrate that the viral capsid functions as a nanoscale container for reverse transcription during HIV-1 infection.
Assuntos
Capsídeo , HIV-1 , Transcrição Reversa , HIV-1/fisiologia , HIV-1/metabolismo , Capsídeo/metabolismo , Humanos , Vírion/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/genética , Montagem de Vírus/fisiologia , Infecções por HIV/virologia , Infecções por HIV/metabolismo , Ácido Fítico/metabolismoRESUMO
Norovirus (NoV) is the main pathogen that causes acute gastroenteritis and brings a heavy socio-economic burden worldwide. In this study, five polysaccharide fractions, labeled pSFP-1-5, were isolated and purified from Sargassum fusiforme (S. fusiforme). In vitro experiments demonstrated that pSFP-5 significantly prevented the binding of type A, B and H histo-blood group antigens (HBGAs) to NoV GII.4 virus-like particles (NoV GII.4 VLPs). In addition, in vivo experiments revealed that pSFP-5 was effective in reducing the accumulation of NoV in oysters, indicating that pSFP-5 could reduce the risk of NoV infection from oyster consumption. The results of transmission electron microscopy showed that the appearance of NoV GII.4 VLPs changed after pSFP-5 treatment, indicating that pSFP-5 may achieve antiviral ability by altering the morphological structure of the viral particles so that they could not bind to HBGAs. The results of the present study indicate that pSFP-5 may be an effective anti-NoV substance and can be used as a potential anti-NoV drug component.
Assuntos
Antígenos de Grupos Sanguíneos , Infecções por Caliciviridae , Norovirus , Polissacarídeos , Sargassum , Norovirus/efeitos dos fármacos , Sargassum/química , Polissacarídeos/farmacologia , Polissacarídeos/química , Polissacarídeos/metabolismo , Animais , Antígenos de Grupos Sanguíneos/metabolismo , Infecções por Caliciviridae/virologia , Infecções por Caliciviridae/tratamento farmacológico , Humanos , Gastroenterite/virologia , Gastroenterite/tratamento farmacológico , Antivirais/farmacologia , Antivirais/química , Ostreidae/virologia , Vírion/metabolismo , Vírion/ultraestrutura , Vírion/efeitos dos fármacos , Algas ComestíveisRESUMO
Nervous necrosis virus (NNV) is a highly neurotropic virus that poses a persistent threat to the survival of multiple fish species. However, its inimitable neuropathogenesis remains largely elusive. To rummage potential partners germane to the nervous system, we investigated the interaction between red-spotted grouper NNV (RGNNV) and grouper brain by immunoprecipitation coupled with mass spectrometry and discerned Nectin1 as a novel host factor subtly involved in viral early invasion events. Nectin1 was abundant in neural tissues and implicated in the inception of tunnel nanotubes triggered by RGNNV. Its overexpression not only dramatically potentiated the replication dynamics of RGNNV in susceptible cells, but also empowered non-sensitive cells to expeditiously capture free virions within 2 min. This potency was impervious to low temperatures but was dose-dependently suppressed by soluble protein or specific antibody of Nectin1 ectodomain, indicating Nectin1 as an attachment receptor for RGNNV. Mechanistically, efficient hijacking of virions by Nectin1 strictly depended on intricate linkages to different modules of viral capsid protein, especially the direct binding between the IgC1 loop and P-domain. More strikingly, despite abortive proliferation in Nectin1-reconstructed CHSE-214 cells, a non-sensitive cell, RGNNV could gain access to the intracellular compartment by capitalizing on Nectin1, thereby inducing canonical cytoplasmic vacuolation. Altogether, our findings delineate a candidate entrance for RGNNV infiltration into the nervous system, which may shed unprecedented insights into the exploration and elucidation of RGNNV pathogenesis.IMPORTANCENervous necrosis virus (NNV) is one of the most virulent pathogens in the aquaculture industry, which inflicts catastrophic damage to ecology, environment, and economy annually around the world. Nevertheless, its idiosyncratic invasion and latency mechanisms pose enormous hardships to epidemic prevention and control. In this study, deploying grouper brain as a natural screening library, a single-transmembrane glycoprotein, Nectin1, was first identified as an emergent functional receptor for red-spotted grouper NNV (RGNNV) that widely allocated in nervous tissues and directly interacted with viral capsid protein through distinct Ig-like loops to bridge virus-host crosstalk, apprehend free virions, and concomitantly propel viral entry. Our findings illuminate the critical role of Nectin1 in RGNNV attachment and entry and provide a potential target for future clinical intervention strategies in the therapeutic race against RGNNV.
Assuntos
Doenças dos Peixes , Nectinas , Nodaviridae , Infecções por Vírus de RNA , Internalização do Vírus , Animais , Nectinas/metabolismo , Nodaviridae/fisiologia , Doenças dos Peixes/virologia , Doenças dos Peixes/metabolismo , Infecções por Vírus de RNA/virologia , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/veterinária , Replicação Viral , Ligação Viral , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Encéfalo/virologia , Encéfalo/metabolismo , Vírion/metabolismo , Linhagem CelularRESUMO
Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.
Assuntos
Membrana Celular , HIV-1 , Fosfatidilinositol 4,5-Difosfato , Montagem de Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana , Produtos do Gene gag do Vírus da Imunodeficiência Humana , HIV-1/fisiologia , HIV-1/metabolismo , Humanos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Membrana Celular/metabolismo , Membrana Celular/virologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Vírion/metabolismo , Células HeLa , Microscopia/métodosRESUMO
Herpes simplex virus 1 (HSV-1) is an alpha herpesvirus that infects a majority of the world population. The mechanisms and cellular host factors involved in the intracellular transport and exocytosis of HSV-1 particles are not fully understood. To elucidate these late steps in the replication cycle, we developed a live-cell fluorescence microscopy assay of HSV-1 virion intracellular trafficking and exocytosis. This method allows us to track individual virus particles and identify the precise moment and location of particle exocytosis using a pH-sensitive reporter. We show that HSV-1 uses the host cell's post-Golgi secretory pathway during egress. The small GTPase, Rab6, binds to nascent secretory vesicles at the trans-Golgi network and plays important, but non-essential, roles in vesicle traffic and exocytosis at the plasma membrane, therefore making it a useful marker of the Golgi and post-Golgi secretory pathway. We show that HSV-1 particles colocalize with Rab6a in the region of the Golgi, cotraffic with Rab6a to the cell periphery, and undergo exocytosis from Rab6a vesicles. Consistent with previous reports, we find that HSV-1 particles accumulate at preferential egress sites in infected cells. The secretory pathway mediates this preferential/polarized egress, since Rab6a vesicles accumulate near the plasma membrane similarly in uninfected cells. These data suggest that, following particle envelopment, HSV-1 egress follows a pre-existing cellular secretory pathway to exit infected cells rather than novel, virus-induced mechanisms. IMPORTANCE: Herpes simplex virus 1 (HSV-1) infects a majority of people. It establishes a life-long latent infection and occasionally reactivates, typically causing characteristic oral or genital lesions. Rarely in healthy natural hosts, but more commonly in zoonotic infections and in elderly, newborn, or immunocompromised patients, HSV-1 can cause severe herpes encephalitis. The precise cellular mechanisms used by HSV-1 remain an important area of research. In particular, the egress pathways that newly assembled virus particles use to exit from infected cells are unclear. In this study, we used fluorescence microscopy to visualize individual virus particles exiting from cells and found that HSV-1 particles use the pre-existing cellular secretory pathway.
Assuntos
Exocitose , Complexo de Golgi , Herpesvirus Humano 1 , Via Secretória , Liberação de Vírus , Proteínas rab de Ligação ao GTP , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Humanos , Animais , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Células Vero , Rede trans-Golgi/metabolismo , Rede trans-Golgi/virologia , Chlorocebus aethiops , Herpes Simples/virologia , Herpes Simples/metabolismo , Vírion/metabolismo , Células HeLa , Membrana Celular/metabolismo , Membrana Celular/virologiaRESUMO
Virus-like particles (VLPs) have untapped potential for packaging and delivery of macromolecular cargo. To be a broadly useful platform, there needs to be a strategy for attaching macromolecules to the inside or the outside of the VLP with minimal modification of the platform or cargo. Here, we repurpose antiviral compounds that bind to hepatitis B virus (HBV) capsids to create a chemical tag to noncovalently attach cargo to the VLP. Our tag consists of a capsid assembly modulator, HAP13, connected to a linker terminating in maleimide. Our cargo is a green fluorescent protein (GFP) with a single addressable cysteine, a feature that can be engineered in many proteins. The HAP-GFP construct maintained HAP's intrinsic ability to bind HBV capsids and accelerate assembly. We investigated the capacity of HAP-GFP to coassemble with HBV capsid protein and bind to preassembled capsids. HAP-GFP binding was concentration-dependent, sensitive to capsid stability, and dependent on linker length. Long linkers had the greatest activity to bind capsids, while short linkers impeded assembly and damaged intact capsids. In coassembly reactions, >20 HAP-GFP molecules were presented on the outside and inside of the capsid, concentrating the cargo by more than 100-fold compared to bulk solution. We also tested an HAP-GFP with a cleavable linker so that external GFP molecules could be removed, resulting in exclusive internal packaging. These results demonstrate a generalizable strategy for attaching cargo to a VLP, supporting development of HBV as a modular VLP platform.
Assuntos
Capsídeo , Proteínas de Fluorescência Verde , Vírus da Hepatite B , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/química , Capsídeo/química , Capsídeo/metabolismo , Montagem de Vírus , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Vírion/metabolismo , Vírion/química , Propriedades de SuperfícieRESUMO
The invention of next-generation CRISPR/Cas gene editing tools, like base and prime editing, for correction of gene variants causing disease, has created hope for in vivo use in patients leading to wider clinical translation. To realize this potential, delivery vehicles that can ferry gene editing tool kits safely and effectively into specific cell populations or tissues are in great demand. In this review, we describe the development of enveloped retrovirus-derived particles as carriers of "ready-to-work" ribonucleoprotein complexes consisting of Cas9-derived editor proteins and single guide RNAs. We present arguments for adapting viruses for cell-targeted protein delivery and describe the status after a decade-long development period, which has already shown effective editing in primary cells, including T cells and hematopoietic stem cells, and in tissues targeted in vivo, including mouse retina, liver, and brain. Emerging evidence has demonstrated that engineered virus-derived nanoparticles can accommodate both base and prime editors and seems to fertilize a sprouting hope that such particles can be further developed and produced in large scale for therapeutic applications.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Ribonucleoproteínas , Edição de Genes/métodos , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Animais , Vírion/metabolismo , Vírion/genética , Retroviridae/genética , Vetores Genéticos/genética , Terapia Genética/métodos , Técnicas de Transferência de Genes , RNA Guia de Sistemas CRISPR-Cas/genéticaRESUMO
The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression and pH change. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into RAP5-negative endosomes via dynamin-dependent scission. Less than two minutes later, VLP speed increases in parallel with a pH drop below 5, yet these two events are not interrelated. By co-imaging fluorescently labeled nucleocapsid proteins, we show that nucleocapsid release occurs with similar kinetics to VLP acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site affected the rate of VLP internalization, indicating that they do not confer any significant advantages or disadvantages during this process. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells.
Assuntos
COVID-19 , Endossomos , SARS-CoV-2 , Internalização do Vírus , SARS-CoV-2/fisiologia , SARS-CoV-2/metabolismo , Humanos , Cinética , COVID-19/virologia , COVID-19/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Endocitose , Animais , Concentração de Íons de Hidrogênio , Chlorocebus aethiops , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Membrana Celular/metabolismo , Membrana Celular/virologia , Vírion/metabolismoRESUMO
Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.
Assuntos
Vírus BK , Vesículas Extracelulares , Infecções por Polyomavirus , Eliminação de Partículas Virais , Vírus BK/fisiologia , Vírus BK/metabolismo , Vírus BK/genética , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virologia , Infecções por Polyomavirus/virologia , Infecções por Polyomavirus/metabolismo , Replicação Viral , Transplante de Rim , Vírion/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Núcleo Celular/metabolismo , Montagem de Vírus , Infecções Tumorais por Vírus/virologia , Infecções Tumorais por Vírus/metabolismo , Transformação Celular Viral , Masculino , AnimaisRESUMO
Recombinant adeno-associated viruses (rAAVs) have emerged as important tools for gene therapy and, more recently, vaccine development. Nonetheless, manufacturing can be costly and time-consuming, emphasizing the importance of alternative production platforms. We investigate the potential of E. coli-based cell-free protein synthesis (CFPS) to produce recombinant AAV5 virus-like particles (VLPs). AAV5 virus protein 3 (VP3) constructs, both with and without Strep-tag II, were expressed with CFPS. Lower reaction temperatures resulted in increased solubility, with the untagged variant containing nearly 90% more soluble VLP VP3 protein at 18 °C than at 37 °C. Affinity chromatography of N-terminally Strep(II)-tagged VP3 enabled successful isolation with minimal processing. DLS and TEM confirmed the presence of â¼20 nm particles. Furthermore, the N-terminally tagged AAV5 VP3 VLPs were biologically active, successfully internalizing into HeLa cells. This study describes an innovative approach to AAV VLP production using E. coli-based CFPS, demonstrating its potential for rapid and biologically active AAV VLP synthesis.
Assuntos
Sistema Livre de Células , Dependovirus , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Dependovirus/genética , Humanos , Células HeLa , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/biossíntese , Biossíntese de Proteínas , Vírion/genética , Vírion/metabolismoRESUMO
Betacoronaviruses encode a conserved accessory gene within the +1 open reading frame (ORF) of nucleocapsid called the internal N gene. This gene is referred to as "I" for mouse hepatitis virus (MHV), ORF9b for severe acute respiratory CoV (SARS-CoV) and SARS-CoV-2, and ORF8b for Middle East respiratory syndrome CoV (MERS-CoV). Previous studies have shown ORF8b and ORF9b have immunoevasive properties, while the only known information for MHV I is its localization within the virion of the hepatotropic/neurotropic A59 strain of MHV. Whether MHV I is an innate immune antagonist or has other functions has not been evaluated. In this report, we show that the I protein of the neurotropic JHM strain of MHV (JHMV) lacks a N terminal domain present in other MHV strains, has immunoevasive properties, and is a component of the virion. Genetic deletion of JHMV I (rJHMVIΔ57-137) resulted in a highly attenuated virus both in vitro and in vivo that displayed a post RNA replication/transcription defect that ultimately resulted in fewer infectious virions packaged compared with wild-type virus. This phenotype was only seen for rJHMVIΔ57-137, suggesting the structural changes predicted for A59 I altered its function, as genetic deletion of A59 I did not change viral replication or pathogenicity. Together, these data show that JHMV I both acts as a mild innate immune antagonist and aids in viral assembly and infectious virus production, and suggest that the internal N proteins from different betacoronaviruses have both common and virus strain-specific properties.IMPORTANCECoV accessory genes are largely studied in overexpression assays and have been identified as innate immune antagonists. However, functions identified after overexpression are often not confirmed in the infected animal host. Furthermore, some accessory proteins are components of the CoV virion, but their role in viral replication and release remains unclear. Here, we utilized reverse genetics to abrogate expression of a conserved CoV accessory gene, the internal N ("I") gene, of the neurotropic JHMV strain of MHV and found that loss of the I gene resulted in a post replication defect that reduced virion assembly and ultimately infectious virus production, while also increasing some inflammatory molecule expression. Thus, the JHMV I protein has roles in virion assembly that were previously underappreciated and in immunoevasion.
Assuntos
Vírus da Hepatite Murina , Proteínas Virais , Replicação Viral , Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/patogenicidade , Vírus da Hepatite Murina/imunologia , Vírus da Hepatite Murina/fisiologia , Animais , Camundongos , Virulência , Proteínas Virais/metabolismo , Proteínas Virais/genética , Vírion/metabolismo , Imunidade Inata , Infecções por Coronavirus/virologia , Infecções por Coronavirus/imunologia , Linhagem Celular , Fases de Leitura Aberta , HumanosRESUMO
Establishing a nonproductive, quiescent infection within monocytes is essential for the spread of human cytomegalovirus (HCMV). We investigated the mechanisms through which HCMV establishes a quiescent infection in monocytes. US28 is a virally encoded G protein-coupled receptor (GPCR) that is essential for silent infections within cells of the myeloid lineage. We found that preformed US28 was rapidly delivered to monocytes by HCMV viral particles, whereas the de novo synthesis of US28 was delayed for several days. A recombinant mutant virus lacking US28 (US28Δ) was unable to establish a quiescent infection, resulting in a fully productive lytic infection able to produce progeny virus. Infection with US28Δ HCMV resulted in the phosphorylation of the serine and threonine kinase Akt at Ser473 and Thr308, in contrast with the phosphorylation of Akt only at Ser473 after WT viral infection. Inhibiting the dual phosphorylation of Akt prevented the lytic replication of US28Δ, and ectopic expression of a constitutively phosphorylated Akt variant triggered lytic replication of wild-type HCMV. Mechanistically, we found that US28 was necessary and sufficient to attenuate epidermal growth factor receptor (EGFR) signaling induced during the entry of WT virus, which led to the site-specific phosphorylation of Akt at Ser473. Thus, particle-delivered US28 fine-tunes Akt activity by limiting HCMV-induced EGFR activation during viral entry, enabling quiescent infection in monocytes.
Assuntos
Citomegalovirus , Receptores ErbB , Monócitos , Proteínas Proto-Oncogênicas c-akt , Proteínas Virais , Replicação Viral , Citomegalovirus/fisiologia , Citomegalovirus/genética , Citomegalovirus/metabolismo , Humanos , Monócitos/virologia , Monócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Fosforilação , Proteínas Virais/metabolismo , Proteínas Virais/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Vírion/metabolismo , Vírion/genética , Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/genética , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/genética , Transdução de SinaisRESUMO
Bovine adenovirus (BAdV)-3 genome encodes a 26 kDa core protein designated as protein VII, which localizes to the nucleus/nucleolus. The requirement of a protein VII-complementing cell line for the replication of VII-deleted BAdV-3 suggests that protein VII is required for the production of infectious progeny virions. An analysis of the BAV.VIId+ virus (only phenotypically positive for protein VII) detected no noticeable differences in the expression and incorporation of viral proteins in the virions. Moreover, protein VII does not appear to be essential for the formation of mature BAV.VIId+. However, protein VII appeared to be required for the efficient assembly of mature BAV.VIId- virions. An analysis of the BAV.VIId- virus (genotypically and phenotypically negative for protein VII) in non-complementing cells detected the inefficient release of virions from endosomes, which affected the expression of viral proteins or DNA replication. Moreover, the absence of protein VII altered the proteolytic cleavage of protein VI of BAV.VIId-. Our results suggest that BAdV-3 protein VII appears to be required for efficient production of mature virions. Moreover, the absence of protein VII produces non-infectious BAdV-3 by altering the release of BAdV-3 from endosomes/vesicles.
Assuntos
Mastadenovirus , Vírion , Replicação Viral , Animais , Vírion/metabolismo , Vírion/genética , Bovinos , Mastadenovirus/genética , Mastadenovirus/fisiologia , Mastadenovirus/metabolismo , Linhagem Celular , Proteínas Virais/metabolismo , Proteínas Virais/genética , Montagem de Vírus , Proteínas do Core Viral/metabolismo , Proteínas do Core Viral/genética , Replicação do DNARESUMO
T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM-1 has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production. IMPORTANCE: Chikungunya virus (CHIKV) is an enveloped alphavirus transmitted by the bites of infectious mosquitoes. Infection with CHIKV results in the development of fever, joint pain, and arthralgia that can become chronic and last for months after infection. Prevention of this disease is still highly focused on vector control strategies. In December 2023, a new live attenuated vaccine against CHIKV was approved by the FDA. We aimed to study the cellular factors involved in CHIKV release, to better understand CHIKV's ability to efficiently infect and spread among a wide variety of cell lines. We found that TIM-1 receptors can significantly abrogate CHIKV's ability to efficiently exit infected cells. This information can be beneficial for maximizing viral particle production in laboratory settings and during vaccine manufacturing.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Receptor Celular 1 do Vírus da Hepatite A , Fosfatidilserinas , Liberação de Vírus , Vírus Chikungunya/fisiologia , Vírus Chikungunya/metabolismo , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Humanos , Fosfatidilserinas/metabolismo , Febre de Chikungunya/virologia , Febre de Chikungunya/metabolismo , Células HEK293 , Internalização do Vírus , Animais , Envelope Viral/metabolismo , Linhagem Celular , Vírion/metabolismo , Receptores Virais/metabolismoRESUMO
Virus-encoded microRNAs (miRNAs) exert diverse regulatory roles in the biological processes of both viruses and hosts. This study delves into the functions of AcMNPV-miR-2, an early miRNA encoded by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). AcMNPV-miR-2 targets viral early genes ac28 (lef-6), ac37 (lef-11), ac49, and ac63. Overexpression of AcMNPV-miR-2 leads to reduced production of infectious budded virions (BVs) and diminished viral DNA replication. Delayed polyhedron formation was observed through light and transmission electron microscopy, and the larval lifespan extended in oral infection assays. Moreover, the mRNA expression levels of two Lepidoptera-specific immune-related proteins, Gloverin and Spod-11-tox, significantly decreased. These findings indicate that AcMNPV-miR-2 restrains viral load, reducing host immune sensitivity. This beneficial effect enables the virus to combat host defense mechanisms and reside within the host for an extended duration. IMPORTANCE: Virus-encoded miRNAs have been extensively studied for their pivotal roles in finetuning viral infections. Baculoviruses, highly pathogenic in insects, remain underexplored concerning their encoded miRNAs. Previous reports outlined three AcMNPV-encoded miRNAs, AcMNPV-miR-1, -miR-3, and -miR-4. This study delves into the functions of another AcMNPV-encoded miRNA, AcMNPV-miR-2 (Ac-miR-2). Through a comprehensive analysis of target gene expression, the impact on larvae, and variations in host immune-related gene expression, we elucidate a functional pathway for Ac-miR-2. This miRNA suppresses viral load and infectivity and prolongs lifespans of infected larva by downregulating specific viral early genes and host immune-related genes. These mechanisms ultimately serve the virus's primary goal of enhanced propagation. Our study significantly contributes to understanding of the intricate regulatory mechanisms of virus-encoded miRNAs in baculovirus infections.